Optical measuring device, measurement method and evaluation method

Abstract

The application relates to an optical measuring device (100) for detecting a spatial distribution (17) of scattered light (11) and to corresponding measurement and evaluation methods. The measuring device (100) comprises: a sample-holding unit (6) for receiving a microfluidic channel (14) for fluidically guiding a sample which comprises sample particles (16), for example cells, embedded in particular in suspended fluid droplets (15); a light source unit (110) which is designed to illuminate the sample with an illumination light (9) such that some of the illumination light (9) can be scattered on the sample for generating the scattered light (11); a detector matrix (8) for detecting the spatial distribution (17) of the scattered light (11); and an apodisation filter (7) which is arranged between the sample and the detector matrix (8), has a preferably radially symmetrical apodisation layer and is designed to attenuate some of the scattered light (11).

Description

[0001] Optical measuring device, measuring method and evaluation method

[0002] The application relates to optical measuring devices for detecting a spatial distribution of scattered light, wherein the spatial distribution provides information in particular about the angular dependence of scattered light from a sample.

[0003] Light. The application further relates to measurement methods using optical measuring devices according to the application and evaluation methods for corresponding measurement data. The subject matter of the application is particularly advantageous in the field of detecting and analyzing microbial, particularly bacterial, cell samples.

[0004] For example, this object can be used in the food industry to test food and drinking water safety (detection of pathogens), in biomedicine for the rapid diagnosis of pathogens or in pharmacy, for example to evaluate the effect of antibiotics and other medications, as well as the rapid detection of possible antibiotic resistance.

[0005] Conventional detection and analysis methods for microbial, particularly bacterial, cell samples (application examples: detection of pathogenic germs, evaluation of the effect of antibiotics, etc.) are often based on cell cultivation, whereby significant results can usually only be determined after pronounced cell growth of several hours to days (formation of cell colonies).

[0006] Conventional methods include DNA (deoxyribonucleic acid)-based assessment of microbial samples, which, however, are based on genotypic predictions and thus may have a high degree of uncertainty, as they do not necessarily reflect the actual phenotypic characteristics (structural properties, reaction behavior to various pharmaceuticals).

[0007] On the other hand, imaging microscopic techniques are widely used to evaluate microbial cell samples. While these techniques often have very good lateral resolution, even the characterization of smaller sample volumes (with depths of a few micrometers) requires confocal measurement approaches combined with vertical sample scanning. At the same time, sensitive detection is usually only achievable with contrast-enhancing dyes or fluorescent substances, which requires intervention in the biological system.

[0008] In addition, there are already scattered light-based approaches for analyzing biological cells, such as flow cytometry. Here, individual scattering directions (usually two measured values) are recorded, allowing different biological cells to be sorted according to significantly different properties (size, morphology). This allows blood cells, for example, to be classified very accurately. However, microbial cells exhibit significantly fewer structural differences in comparison and can therefore often only be roughly assessed using this method.

[0009] A light scattering technique for the investigation and detection of microbial (specifically bacterial) cells is described in the document "Using Scattering to Identify Bacterial Pathogens," Optics & Photonics News, vol. 22, no. 10, p. 20, 2011, by JP Robinson, BP Rajwa, E. Bae, V. Patsekin, AM Roumani, AK Bhunia, JE Dietz, VJ Davisson, MM Undar, J. Thomas, and E. . Hirleman. However, the measurement and analysis here are performed on already developed cell cultures with diameters in the range of 1 mm, thus targeting the specifics of the cell culture rather than a few individual cells. This also results in comparatively longer growth times.

[0010] A scattered light method (for surface measurements) and corresponding arrangements are also described in the document DE 10 2009 036 383 B3.

[0011] There is an urgent need for sensitive and therefore rapid analysis technologies for the evaluation of microbiological cell samples (e.g. cell type) as well as cell growth (cell number / concentration) even with small sample volumes, ideally from just a few individual cells.

[0012] To solve the problem, optical measuring devices, measuring methods and evaluation methods are proposed according to the patent claims and according to further examples described below.

[0013] An optical measuring device of the proposed type is configured to detect a spatial distribution of scattered light and comprises a sample holding unit for receiving a microfluidic channel for fluidically conducting a sample, which in particular comprises sample particles, for example cells, embedded in suspended fluid droplets, a light source unit configured to illuminate the sample with an illumination light such that a portion of the illumination light is scattered by the sample to generate the scattered light, a detector matrix for detecting the spatial distribution of the scattered light, and an apodization filter arranged between the sample and the detector matrix, configured to attenuate a portion of the scattered light.

[0014] This provides a scattered light measurement system that is compact, fast, and robust, yet highly sensitive. In particular, it enables the combination of fast yet highly sensitive detection and analysis of small (volume or cell number) microbiological cell samples.

[0015] The apodization filter compensates for the expected measurement dynamics, thus enabling particularly fast acquisition of a wide intensity dynamic range, such as that typically encountered in scattered light distributions. Conventional methods achieve this, for example, through a sequence of multiple measurements with different exposure times. By using the apodization filter, however, this can be achieved with a single measurement.

[0016] The apodization layer is preferably radially symmetric, i.e., preferably has a rotationally symmetric transmission distribution. The apodization layer can, for example, have a rotationally symmetric logarithmic transmission distribution. The apodization filter can have an anti-reflective coating to achieve particularly low back reflection of the filter, for example, less than 0.1%, and / or reduced self-scattering within an angular range of approximately ±60°. Additional measures such as apertures can be taken to further suppress irrelevant stray light components.

[0017] The sample holding unit designed to accommodate the microfluidic channel enables rapid measurements with high throughput and small sample volumes—particularly in conjunction with samples containing sample particles embedded in suspended fluid droplets. This also results in low consumption of reagents and sample material. The detector matrix can be used to capture the forward-directed two-dimensional scattered light distribution of the sample as a function of the scattering direction or angle. The detector matrix can be formed, for example, by a CMOS sensor, a CCD sensor, a planar arrangement of highly sensitive detectors (e.g., a photodiode array), etc.

[0018] The measuring device may further comprise a beam trap arranged between the sample and the detector array for absorbing a portion of the illumination light transmitted as a direct beam to the sample. This can reduce the interference of the direct beam, for example, by saturating areas of the detector array.

[0019] The light source unit can comprise a focusing unit for focusing the illumination light in or near the sample and preferably a spatial filter, in particular a pinhole. By focusing in or near the sample, the spot size in the focus can be selected, for example, to match the size of the microfluidic channel and fluid droplet. This allows corresponding interference patterns (e.g., stray light rings from the droplet) to be filtered out in the subsequent data analysis. The spatial filter can be used to optically clean the illumination light beam. The light source unit preferably comprises a laser light source.

[0020] The measuring device may further comprise the microfluidic channel, wherein the microfluidic channel and / or a substrate supporting the microfluidic channel comprises a mask layer for absorbing and / or reflecting back-reflected light components, wherein the mask layer is configured to transmit the scattered light and the direct beam. Interfering influences of back-reflected light components on the measurement can thus be reduced.

[0021] The measuring device may further comprise: a trigger circuit configured to provide a trigger signal upon passage of a fluid droplet of the sample through the illumination light, such that the trigger signal triggers the capture of a single image by means of the detector matrix.

[0022] A measuring method of the proposed type is suitable for detecting a scattered light distribution, in particular using an optical measuring device of the proposed type. The measuring method comprises:

[0023] Illuminate the sample with the illumination light,

[0024] Detecting the spatial distribution of scattered light using the detector matrix.

[0025] It can be provided that the sample comprises cells, in particular bacterial cells, embedded in fluid droplets, wherein the fluid droplets are guided individually through the illumination light by means of the microfluidic channel, so that the respectively detected spatial distribution of the scattered light enables a determination of a number and / or at least one morphological parameter of the cells contained in each fluid droplet.

[0026] It can be provided that the illumination light is focused by means of the focusing unit in or near the sample such that the ratio of the diameter of the resulting illumination region to the diameter of the fluid droplets is in the range of 0.2 to 1.2, preferably in the range of 0.4 to 0.8. For example, a diameter of the illumination region of less than 100 pm at the sample position can be achieved with the lowest possible divergence, for example, an aperture angle of less than ±4°.

[0027] It can be provided that the cells are removed from a cell culture at at least two different times and embedded in the fluid droplets, so that a time course of the number and / or of the at least one morphological parameter of the cells contained in the fluid droplets can be determined.

[0028] It can be provided that the fluid droplets with the embedded cells are guided through the illumination light by means of the microfluidic channel at at least two different times, so that a time course of the number and / or of the at least one morphological parameter of the cells contained in the fluid droplets can be determined.

[0029] The embedding of fluid droplets at different times and the guiding of the fluid droplets through the illumination light at different times can be combined and / or performed in parallel. This allows for a combined time course and / or a comparison of time courses.

[0030] The measurement method may further comprise: detecting a reference scattered light distribution resulting from illuminating a reference sample with the illumination light, wherein the reference sample comprises sample particles in free suspension and / or immobilized sample particles and / or suspended fluid droplets without sample particles and / or suspended fluid droplets with a known concentration of the sample particles and / or a fluid without the suspended fluid droplets and / or a diffuser. Such a diffuser may, for example, be or comprise a white glass diffuser and / or a structured glass diffuser.

[0031] Recorded scattered light distributions result from the coherent superposition of scattered light effects on the sample support structure (e.g., slide, microfluidic chip), the surrounding medium of the cells (e.g., nutrient medium), and the microorganisms under investigation with their structural properties: shape, size, morphology, cell aggregation, and cell count. For example, when assessing the cell count and / or cell type in microfluidically guided nutrient droplets, the recorded scattered light distribution is influenced by the microfluidic chip, the droplets consisting of nutrient medium, and the cells they contain.

[0032] Suitable measurement and evaluation methods aim to separate these influencing factors. For this purpose, a comparative measurement (blank measurement) can be performed using a reference sample, such as one of the species mentioned above, and compared with the measurements on the biological material. This allows interfering influences to be separated.

[0033] In order to filter out the relevant information from the remaining complex scattered light information of the sample, various data evaluation methods can then be applied individually or in combination, such as:

[0034] A) Integration of angle-resolved scattered light information

[0035] B) Analysis of the slope and shape of the scattered light distributions C) Structure and pattern analysis in the frequency domain

[0036] D) Neural network trained by a large number of recorded scattered light distributions.

[0037] An evaluation method of the proposed type is suitable for measurement data acquired by means of a measurement method of the proposed type and / or using a measuring device of the proposed type.

[0038] Such an evaluation method may comprise: segmenting the detected spatial distribution of the scattered light to extract a plurality of structural features of the spatial distribution by means of a trained model, in particular based on a convolutional neural network.

[0039] The evaluation method may further comprise: processing the extracted structural features by means of a principal component analysis to determine a characteristic set of parameters of the spatial distribution.

[0040] The evaluation method may further comprise: quantifying a sample parameter, in particular an optical density and / or a sample particle concentration and / or a morphological parameter, based on the characteristic parameter set.

[0041] Another proposed evaluation method comprises quantifying a sample parameter, in particular an optical density and / or a sample particle concentration and / or a morphological parameter, using a trained model, in particular based on a convolutional neural network. This method step can also further develop the previously described method, or vice versa.

[0042] Through appropriate calibration, traceable, device-independent, and thus comparable, angle-resolved scattered light data can be generated, for example, in the form of the ISO standardized measurement parameter ARS (ISO 19986). The calibration procedures provided for this purpose can include both a general calibration of illumination power and detector solid angle, as well as a calibration of apodization. For example, the intensity distribution of a known, strongly scattering sample (such as a diffuser of the type mentioned above) can be recorded.

[0043] Examples of the subject matter of the application are explained below with reference to drawings. These show, in schematic and simplified form,

[0044] FIG. 1 shows a beam path diagram of an optical measuring device,

[0045] FIG. 2 shows part of the optical measuring device according to FIG. 1, illustrating aspects of a measuring method.

[0046] Recurring and similar features are identified in the drawings with identical alphanumeric reference symbols. Reference symbols already shown in other drawings may be partially omitted.

[0047] The optical measuring device 100 shown in FIG. 1 and partially in FIG. 2 comprises a sample holding unit 6 for receiving a microfluidic channel 14 for fluidically conducting a sample, which in particular comprises sample particles 16, for example cells, embedded in suspended fluid droplets 15, a light source unit 110 configured to illuminate the sample with an illumination light 9 such that a portion of the illumination light 9 is scattered by the sample to generate the scattered light 11 and a portion of the illumination light is transmitted as a direct beam 10, a CMOS sensor as a detector matrix 8 for detecting a spatial distribution 17 of the scattered light, an apodization filter 7 arranged between the sample and the detector matrix 8 with a preferably radially symmetric apodization layer, configured to attenuate a portion of the scattered light 11.

[0048] The measuring device 100 further comprises: a beam trap 12 arranged between the sample and the detector matrix 8 for absorbing the direct beam.

[0049] The light source unit 110 comprises a focusing unit 120 with a second focusing lens 5 for focusing the illumination light 9 in or near the sample, and a spatial filter with a first focusing lens 2 and a pinhole 3. The illumination light can be shaped or limited by additional apertures (here, for example, aperture 4). The light source unit 110 comprises a laser light source 1.

[0050] The measuring device 100 comprises a photodiode 13 for detecting a portion of the scattered light when a fluid droplet 15 passes through the illumination light 9, as well as a trigger circuit configured to provide a trigger signal 18 based on a corresponding passage signal 19, so that the trigger signal triggers the capture of an individual image by means of the detector matrix 8.

[0051] In an exemplary measurement method, the sample comprises bacterial cells embedded in fluid droplets 15 as sample particles 16, wherein the fluid droplets 15 are guided individually through the illumination light 9 by means of the microfluidic channel 14, so that the respectively detected spatial distribution 17 of the scattered light 11 enables a determination of a number and / or at least one morphological parameter of the cells contained in each fluid droplet 15 by means of evaluation methods of the type explained above.

[0052] It can be provided that cells are taken from a cell culture at at least two different times and embedded in the fluid droplets 15 and / or that the fluid droplets 15 with already embedded cells are guided through the illumination light 9 at at least two different times by means of the microfluidic channel 14, so that by evaluating the respectively recorded spatial distribution 17 of the scattered light 11, a time course of the number and / or of the at least one morphological parameter of the cells contained in the fluid droplets 15 can be determined.

[0053] List of reference symbols:

[0054] 100 Optical measuring device

[0055] 110 Light source unit

[0056] 120 Focusing unit

[0057] 1 laser light source

[0058] 2 first focusing lens

[0059] 3 pinholes

[0060] 4 aperture

[0061] 5 second focusing lens

[0062] 6 Sample holding unit

[0063] 7 apodization filters

[0064] 8 detector matrix

[0065] 9 Illumination light

[0066] 10 direct beam

[0067] 11 Scattered light

[0068] 12 beam trap

[0069] 13 Photodiode

[0070] 14 microfluidic channel

[0071] 15 fluid droplets

[0072] 16 sample particles

[0073] 17 spatial distribution

[0074] 18 Trigger signal

[0075] 19 Passage signal.

Claims

Patent claims 1. An optical measuring device (100) for detecting a spatial distribution (17) of a scattered light (11), comprising a sample holding unit (6) for receiving a microfluidic channel (14) for fluidically conducting a sample, which in particular comprises sample particles (16), for example cells, embedded in suspended fluid droplets (15), a light source unit (110) configured to illuminate the sample with an illuminating light (9) such that a portion of the illuminating light (9) is scattered by the sample to generate the scattered light (11), a detector matrix (8) for detecting the spatial distribution (17) of the scattered light (11), an apodization filter (7) arranged between the sample and the detector matrix (8) with a preferably radially symmetrical apodization layer, configured to attenuate a portion of the scattered light (11).

2. Optical measuring device (100) according to claim 1, further comprising a beam trap (12) arranged between the sample and the detector matrix (8) for absorbing a part of the illumination light (9) transmitted as a direct beam (10) at the sample.

3. Optical measuring device (100) according to one of the preceding claims, wherein the light source unit (110) comprises a focusing unit (120) for focusing the illumination light (9) in or near the sample and preferably a spatial filter, in particular a pinhole (3).

4. Optical measuring device (100) according to one of the preceding claims, further comprising the microfluidic channel (14), wherein the microfluidic channel (14) and / or a substrate supporting the microfluidic channel (14) comprises a mask layer for absorbing and / or reflecting retroreflected light components, wherein the mask layer is configured to transmit the scattered light (11) and the direct beam (10).

5. Optical measuring device (100) according to one of the preceding claims, further comprising a trigger circuit configured to provide a trigger signal (18) upon passage of a fluid droplet (15) of the sample through the illumination light (9), so that the trigger signal (18) triggers the capture of an individual image by means of the detector matrix (8).

6. A measuring method for detecting a scattered light distribution using the optical measuring device (100) according to one of the preceding claims, comprising Illuminating the sample with the illuminating light (9), Detecting the spatial distribution (17) of the scattered light (11) by means of the detector matrix (8).

7. Measuring method according to the preceding claim, wherein the sample comprises cells, in particular bacterial cells, embedded in fluid droplets (15), wherein the fluid droplets (15) are guided individually through the illumination light (9) by means of the microfluidic channel (14), so that the respectively detected spatial distribution (17) of the scattered light (11) enables a determination of a number and / or at least one morphological parameter of the cells contained in each fluid droplet (15).

8. Measuring method according to the preceding claim, insofar as it is dependent on claim 3, wherein the illuminating light (9) is focused by means of the focusing unit (120) in or near the sample such that a ratio of a diameter of the resulting illumination region to a diameter of the fluid droplets (15) is in the range from 0.2 to 1.2, preferably in the range from 0.4 to 0.

8.

9. Measuring method according to one of claims 7 and 8, wherein the cells are removed from a cell culture at at least two different times and embedded in the fluid droplets (15) and / or the fluid droplets (15) with the embedded cells are guided through the illumination light (9) at at least two different times by means of the microfluidic channel (14), so that a time course of the number and / or of the at least one morphological parameter of the cells contained in the fluid droplets (15) can be determined.

10. Measuring method according to one of claims 6 to 9, further comprising Detecting a reference scattered light distribution resulting from illuminating a reference sample with the illumination light (9), wherein the reference sample comprises sample particles (16) in free suspension and / or immobilized sample particles (16) and / or suspended fluid droplets (15) without sample particles (16) and / or suspended fluid droplets (15) with a known concentration of the sample particles (16) and / or a fluid without the suspended fluid droplets (15) and / or a diffuser.

11. Evaluation method for measurement data acquired by means of the measurement method according to one of claims 6 to 10, comprising Segmenting the detected spatial distribution (17) of the scattered light (11) to extract a plurality of structural features of the spatial distribution (17) by means of a trained model, in particular based on a convolutional neural network.

12. Evaluation method according to the preceding claim, further comprising Processing the extracted structural features using principal component analysis to determine a characteristic set of parameters of the spatial distribution (17).

13. Evaluation method according to the preceding claim, further comprising Quantifying a sample parameter, in particular an optical density and / or a sample particle concentration and / or a morphological parameter, based on the characteristic parameter set.

14. Evaluation method for measurement data acquired by means of the measurement method according to one of claims 6 to 10, comprising Quantifying a sample parameter, in particular an optical density and / or a sample particle concentration and / or a morphological parameter, by means of a trained model, in particular based on a convolutional neural network.

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